1. Field of the Invention
The present invention is related generally to immunosensors, and more specifically to immunosensors for flow-through displacement immunoassays.
2. Description of the Background Art
Immunoassays exist in a variety of formats that utilize the interaction of antibodies with antigens usually including direct binding, competitive and sandwich assay schemes. The continuous flow immunoassay is a unique displacement assay that measures the dissociation of a fluorescently labeled antigen from an antibody bound on a solid support when the antigen flows past the antibody (U.S. Pat. No. 5,183,740, issued Feb. 2, 1993 to Ligler et al., the entirety of which is incorporated herein by reference for all purposes.) The displacement of the labeled antigen is proportional to the quantity of antigen present in the sample. Sensitivity of the assay is dependent on the dissociation constant of the antibody and the probability of antigen-to-antibody interaction in the flow system.
In previous studies on flow immunoassays using columns of packed beads or porous membranes as substrates for the antibody immobilization, the following parameters have been determined to affect signal magnitude and assay sensitivity:
(1) The affinity of the antibody for the antigen must be as high or higher than the affinity of the antibody for the labeled analog under the conditions of operation of the displacement immunoassay.
(2) There is a minimum number of antibody-labeled antigen complexes that must be present in the assay in order to generate a signal. Past this minimum level, increasing the number of antibodies by increasing the amount of substrate or antibody density increases the signal magnitude and the number of assays that can be performed, but may decrease the antigen sensitivity, probably due to rebinding of labeled antigen to immobilized antibody. The minimum detectable amount of displaced labeled antigen is also a function of detector sensitivity.
(3) For each antigen-antibody pair, there seems to be an optimum flow rate which is probably related to the dissociation constant of the antibody. Increasing the flow rate above this level increases spontaneous dissociation of labeled antigen, decreases antigen-antibody interaction time, and, consequently decreases the displacement efficiency (ratio of the number of moles of antigen added vs. number of moles of labeled antigen displaced). Decreasing the flow rate too much results in poor discrimination of the signal from background due to peak broadening. In general, flow rates of 0.1 to 2.0 ml/min are taught with 0.2 to 1 ml/min being preferable (U.S. Pat. No. 5,183,740, infra). If not for an unacceptably low signal to background ratio, low flow rates would be desirable for the detection of low analyte concentrations in small (e.g., one picoliter to ten microliters) samples.
In Wemhoff et al. (Wemhoff, G. A., S. Y. Rabbany, A. W. Kusterbeck, R. A. Ogert, R. Bredehorst, and F.S. Ligler J. Immunol. Methods, 156, 223-230, 1992), the concept of displacement efficiency was introduced as means for comparing assay performance as various parameters were modified. The displacement efficiency is at its maximum when the concentration of antigen added is low relative to the dynamic range for the column being used. The amount of displaced labeled antigen molecules does not exceed the displacement efficiency times the concentration of labeled antigen bound to the immobilized antibody, even when high concentrations of antigen are added. For the packed bed columns, a maximum displacement efficiency of about 0.001 was typical under optimal flow conditions.
U.S. Pat. No. 5,183,740 to Ligler et al. teaches a variety of support media, including capillary tubes. That patent, however, attaches no criticality to the form of the support media or column. Moreover, that patent fails to teach a range of capillary inner diameters and lengths and fails to suggest any relationship between capillary diameter/length and sensitivity.
Finally, obtaining consistent results from sample to sample requires columns that can be manufactured consistently, reliably, and reproducibly. Packing columns with beads is an imprecise process that results in variability among columns.